Cell Fixation by Light-Triggered Release of Glutaraldehyde

Article abstract of DOI:10.1002/anie.201612112

Chemical fixation of living cells for microscopy is commonly achieved by crosslinking of intracellular proteins with dialdehydes prior to examination. We herein report a photocleavable protecting group for glutaraldehyde that results in a light-triggered and membrane-permeable fixative, which is nontoxic prior to photocleavage. Lipophilic ester groups allow for diffusion across the cell membrane and intracellular accumulation after enzymatic hydrolysis. Irradiation with UV light releases glutaraldehyde. The in situ generated fixative crosslinks intracellular proteins and preserves and stabilizes the cell so that it is ready for microscopy. In contrast to conventional glutaraldehyde fixation, tissue autofluorescence does not increase after fixation. Caged glutaraldehyde may in future enable functional experiments on living cells under a light microscope in which events of interest can be stopped in spatially confined volumes at defined time points. Samples with individually stopped events could then later be analyzed in ultrastructural studies.

Full text of DOI:10.1002/anie.201612112

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201612112
German Edition: DOI: 10.1002/ange.201612112
Cell Fixation
Hot Paper
Cell Fixation by Light-Triggered Release of Glutaraldehyde
Korwin M. Schelkle+, Christopher Schmid+, Klaus Yserentant, Markus Bender, Irene Wacker,
Martin Petzoldt, Manuel Hamburger, Dirk-Peter Herten, Richard Wombacher,
Rasmus R. Schrçder, and Uwe H. F. Bunz*
Abstract: Chemical fixation of living cells for microscopy is
commonly achieved by crosslinking of intracellular proteins
with dialdehydes prior to examination. We herein report
a photocleavable protecting group for glutaraldehyde that
results in a light-triggered and membrane-permeable fixative,
which is nontoxic prior to photocleavage. Lipophilic ester
groups allow for diffusion across the cell membrane and
intracellular accumulation after enzymatic hydrolysis. Irradi-
ation with UV light releases glutaraldehyde. The in situ
generated fixative crosslinks intracellular proteins and pre-
serves and stabilizes the cell so that it is ready for microscopy.
In contrast to conventional glutaraldehyde fixation, tissue
autofluorescence does not increase after fixation. Caged
glutaraldehyde may in future enable functional experiments
on living cells under a light microscope in which events of
interest can be stopped in spatially confined volumes at defined
time points. Samples with individually stopped events could
then later be analyzed in ultrastructural studies.
In contrast to conventional fixation with aldehydes, rapid
freezing techniques (e.g., high-pressure freezing) allow for
instantaneous physical fixation. These techniques, however,
require special equipment and manipulation of the sample at
low temperatures (below À1008C).[6,7]
Yet, both fixation methods represent integral approaches
that result in the fixation of the complete sample. Instanta-
neous fixation with the potential for high spatial resolution
would be an important complement to the known fixation
techniques. Our investigation started from the idea to
introduce a photocleavable protected glutaraldehyde into
cells that is activated by light for immediate fixation (cell
fixation by flash photolysis, “Cellfi-flash”).[8] Photocleavable
protecting groups enable the modulation of molecular
processes by light with both temporal and spatial resolution.
Light as an external stimulus easily penetrates microscopy
samples and thus has access to reaction centers that are
otherwise difficult to reach.[9] The fixative is masked with
a photocleavable functional group to block the crosslinking
activity prior to irradiation.[10]
Since the first demonstration by Sabatini and co-workers,
aldehydes have been the most commonly used fixatives for
light and electron microscopy owing to their unrivalled ability
to stabilize the fine structural details of cells and tissues prior
to examination.[1,2] Among them, glutaraldehyde is the
aldehyde of choice because of its high crosslinking reactivity,
allowing superior structural preservation in combination with
fast fixation.[3,4] However, in conventional procedures, fix-
ation with aldehydes is a diffusion-controlled process as the
employed aldehydes have to enter a cell across its membrane,
a process that is not instantaneous. During that time lag, the
chemical fixative can cause severe changes within the cell.[5]
Various photocleavable protecting groups have been
established and used to modulate the localization,[11] inter-
action,[12] and activity[13,14] of biomolecules. The ideal caged
molecule should 1) be noncytotoxic, 2) provide high uncaging
efficiencies, 3) have sufficient solubility in buffered solution,
4) be sufficiently membrane-permeable, and 5) feature
a mechanism for accumulation and trapping of the compound
after getting into the cell. For the design and synthesis of
caged glutaraldehyde, we were inspired by work from the
groups of Gravel and Ni and identified the structural motive
of ortho-nitrophenylethylene glycol as a promising starting
[*] Dr. K. M. Schelkle,[+] M. Sc. M. Bender, Dr. M. Petzoldt,
Dr. M. Hamburger, Prof. Dr. U. H. F. Bunz
Organisch-Chemisches Institut
Dr. R. Wombacher
Institut fꢁr Pharmazie und Molekulare Biotechnologie
Ruprecht-Karls-Universitꢀt Heidelberg
Ruprecht-Karls-Universitꢀt Heidelberg
Im Neuenheimer Feld 364, 69120 Heidelberg (Germany)
Im Neuenheimer Feld 270, 69120 Heidelberg (Germany)
E-mail: uwe.bunz@oci.uni-heidelberg.de
C. Schmid,[+] M. Sc. K. Yserentant, Dr. I. Wacker,
Prof. Dr. R. R. Schrçder, Prof. Dr. U. H. F. Bunz
Center of Advanced Materials
Ruprecht-Karls-Universitꢀt Heidelberg
Im Neuenheimer Feld 225, 69120 Heidelberg (Germany)
Dr. K. M. Schelkle,[+] Dr. M. Petzoldt, Dr. M. Hamburger
M. Sc. K. Yserentant, Prof. Dr. D.-P. Herten
CellNetworks, Single Molecule Spectroscopy
Ruprecht-Karls-Universitꢀt Heidelberg
Im Neuenheimer Feld 267, 69120 Heidelberg (Germany)
Prof. Dr. R. R. Schrçder
CellNetworks, Cryo Electron Microscopy
Universitꢀtsklinikum Heidelberg
Im Neuenheimer Feld 267, 69120 Heidelberg (Germany)
[+] These authors contributed equally to this work.
InnovationLab GmbH
Speyerer Strasse 4, 69115 Heidelberg (Germany)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
http://dx.doi.org/10.1002/anie.201612112.
M. Sc. K. Yserentant, Prof. Dr. D.-P. Herten
Physikalisch-Chemisches Institut
Ruprecht-Karls-Universitꢀt Heidelberg
Im Neuenheimer Feld 229, 69120 Heidelberg (Germany)
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chemical actinometry.[17] The course of the photolysis reaction
was followed by ultra-performance liquid chromatography
mass spectrometry (UPLC-MS; Figure 1). Along with the
decrease in the amount of caged-GA 8, an increase in the
amount of cleavage product 10 and the monoprotected
glutaraldehyde 9 was observed (see the Supporting Informa-
tion for the corresponding spectra). Acetoxymethyl (AM)
esters are commonly used when membrane permeability and
intracellular accumulation are desired.[18] Based on studies by
Thodi and co-workers,[19] we investigated whether our ethyl
esters are also cleaved by esterases. Assuming that caged-GA
8 is lipophilic enough to permeate the cell membrane,
intracellular cleavage of the ethyl esters would generate
carboxylates, which are anionic at physiological pH and not
membrane-permeable anymore. Thus hydrolyzed caged-GA
8 would be trapped within the cell, ensuring continuous
intracellular accumulation. To probe whether ethyl esters are
cleaved by esterases, we used porcine liver esterase and
lipase B extracted from Candida antarctica as two represen-
tative esterases at various enzyme/substrate concentrations,
and the reaction mixtures were subsequently subjected to
UPLC-MS analysis. Complete consumption of the tetracar-
boxylic ester was observed within 1 h for all enzyme/substrate
ratios. For porcine liver esterase, mainly tri- and tetracarbox-
ylic acids were detected, while lipase B of Candida antarctica
exclusively hydrolyzed until the dicarboxylic acid stage
(Figure 2; see the Supporting Information for the correspond-
ing spectra). The cytotoxicity of caged-GA 8 was tested first
by live/dead staining. The presence of only green fluorescent
cells demonstrated that cells survive treatment with the
compound for several hours. As the fluorescence intensity of
the treated cells after incubation with caged-GA 8 was lower
than that of nonincubated samples, we assume that caged-GA
8 bearing two nitro groups, which could be responsible for the
observed fluorescence quenching, is membrane-permeable
without apparent cytotoxic side effects (see the Supporting
Information).[20]
Scheme 1. Synthesis of caged glutaraldehyde 8. p-TsOH=para-toluene-
sulfonic acid, TFA=trifluoroacetic acid.
point.[15,16] The synthesis of caged glutaraldehyde 8 is shown in
Scheme 1. The photocleavable protecting group 7 was pre-
pared from 3,4-dihydroxyacetophenone (1) in six steps:
O-alkylation with ethyl bromoacetate, nitration, a-bromina-
tion, acetylation of the a-bromoketone, a-hydroxylation, and
subsequent reduction of hydroxyketone 6 afforded diol 7. In
the last step, acid-catalyzed addition of diol 7 to glutaralde-
hyde yielded caged glutaraldehyde 8 (caged-GA 8) in 14%
overall yield.
In aqueous phosphate
buffer
solution
(0.1m,
pH 7.4), caged-GA 8 dis-
plays two UV absorption
peaks at l = 300 nm and
l = 342 nm (e300 nm = 1.06 ꢀ
103 mÀ1 cmÀ1 and e342 nm
=
1.03 ꢀ 103 mÀ1 cmÀ1, respec-
tively; see the Supporting
Information for the corre-
sponding spectra). Photol-
ysis of caged-GA 8 in aque-
ous
phosphate
buffer
(0.1m, pH 7.4) upon irradi-
ation at l = 300 nm in
a Rayonet RPR-200 photo-
reactor was fast and pro-
ceeded with
efficiency of f = 0.30,
a quantum
Figure 1. A) UPLC-MS elugram for the photolysis of caged glutaraldehyde 8 after irradiation at l=300 nm for
20 s. B) Decrease in the concentration of caged-GA 8 during photolysis. The corresponding HPLC traces can
which was determined by be found in the Supporting Information.
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Figure 2. Enzymatic ester hydrolysis of caged-GA 8 at different
enzyme/substrate ratios by porcine liver esterase. Complete consump-
tion of the tetraester was observed. The data for enzymatic ester
hydrolysis by lipase B extracted from Candida antarctica and the
corresponding HPLC traces can be found in the Supporting Informa-
tion.
Figure 3. A) Kymographs of the mitochondrial motion in Hela cells
and B) mitochondrial velocity in HeLa cells exposed to 250 mm (blue),
125 mm (green), or 65 mm (orange) of caged-GA 8 or 2.5% DMSO
solution (violet) before (left) and after (right) irradiation at l=300 nm.
The velocities are given as the average of six mitochondrial traces with
the standard deviations shown as error bars.
To assess the applicability of caged-GA 8 as a cellular
fixative, we tracked the movement of fluorescently stained
mitochondria in living cells by fluorescence microscopy. After
overnight incubation with caged-GA 8, excess (not cell-
attached or not cell-internalized) caged-GA 8 was washed out
(see the Supporting Information). After washing, mitochon-
drial movement was recorded before and after uncaging.
Uncaging was performed by irradiation with UV light at l =
300 nm for 2 min in a homemade reaction chamber bearing
eight lamps of a Rayonet RPR-200 photoreactor. At all
concentrations (250 mm, 125 mm, 65 mm), uncaging stopped the
mitochondrial movement (see Figure 3 and Movie S1) while
the mitochondrial motility of untreated control cells was
unaffected by exposure to UV light (Figure 3). The finding
that both the mitochondrial morphology and motility are
unaffected by 12 h exposure to caged-GA 8 is an additional
indication of the low cytotoxicity of the uncleaved compound
as mitochondria tend to fractionate under stress.
We compared the fixation kinetics of caged-GA 8 (con-
centrations as indicated above) and commonly employed
aldehydes, namely glutaraldehyde (GA) and paraformalde-
hyde (PFA), at different concentrations by monitoring the
decrease in mitochondrial motility with time. Mitochondria
were instantaneously (in less than 1 min) immobilized by
PFA. Rapid fixation using glutaraldehyde was observed only
at a minimum concentration of 200 mm. At the lowest GA
concentration, equivalent to the lowest concentration of
caged-GA 8, mitochondrial motility stopped only after
20 min. Caged-GA 8 instantly stalled mitochondrial move-
ment upon its uncaging (see Movie S1) at all concentrations
tested. Based on these experiments, caged-GA 8 is on par
with PFA and superior to glutaraldehyde at the lowest
concentrations tested (Figure 4). 200 mm GA or 1.2m PFA,
concentrations routinely used in cell biology, increase cellular
autofluorescence as indicated by the lower contrast after
fixation with PFA or GA (Figure 5D,F). The increase in
autofluorescence for l = 640 nm (red) excitation is about 40–
Figure 4. Decrease in the mitochondrial motility of HeLa cells exposed
to either caged-GA 8 (250 mm, 125 mm, 65 mm) and UV treatment, GA
(200 mm, 200 mm, 65 mm), or PFA (1.2m, 120 mm, 65 mm). For each
time span, the motion of six mitochondrial traces was analyzed per
fixative concentration. * Concentrations of GA and PFA routinely used
in cell biology.
60% with PFA, and amounts to a factor of 20 for GA
(Figure 5G). In contrast, caged-GA 8 did not elevate cellular
autofluorescence after uncaging (Figure 5G).
Light microscopy revealed that cells exposed to caged-
GA 8 are covered with micellar structures (see Figure 5 and
the Supporting Information). We analyzed their size by
negative-stain electron microscopy, which revealed that the
size of the micellar structures ranged from about 500 nm to
almost 6 mm (see the Supporting Information).
In conclusion, we have synthesized a caged glutaralde-
hyde derivative that is efficiently uncaged upon exposure to
UV light and rapidly fixes mammalian cells. In contrast to
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groups that can be cleaved off by light of longer wavelengths
or two-photon absorption to avoid the use of potentially
harmful UV light. In addition, we will further investigate the
uptake mechanism of the caged glutaraldehyde derivative 8
across the cell membrane by selectively inhibiting the cellular
endocytic machinery.
Acknowledgements
This work was financially supported within the leading-edge
cluster “Forum Organic Electronics” as part of the High-Tech
Strategy for Germany of the Federal Ministry of Education
and Research (FKZ 13N11701) and the collaborative con-
sortium “MorphiQuant-3D” (FKZ 13GW0044). K.M.S.
thanks the Chemical Industry Fund of the German Chemical
Industry Association (VCI) for a scholarship.
Conflict of interest
The authors declare no conflict of interest.
Keywords: cell fixation · glutaraldehyde · microscopy ·
mitochondrial motion · photocleavage
Figure 5. Cellular autofluorescence in HeLa cells stained with Mito-
Tracker Deep Red before (A,C,E) and after fixation using B) 250 mm
caged-GA 8 and 2’ UV treatment, D) 1.2m (4%) PFA, and F) 200 mm
(2%) GA. G) Autofluorescence in unstained HeLa cells upon excitation
in the blue/green (488 nm, light bars), green/orange (561 nm, gray
bars), and red (640 nm, dark bars) spectral region. The intensities per
cell were normalized against the autofluorescence of live untreated
cells. At least 20 cells were quantified per condition; error bars
represent one standard deviation. The elevated autofluorescence in the
blue/green spectral channel for 8 is due to photolabile caging groups.
Please note that (A)–(F) show the mixed signal of autofluorescence
and MitoTracker (640 nm) while (G) indicates the contribution of the
autofluorescence of unstained cells. Based on these data, we con-
cluded that uncaging 8 does not generate autofluorescence.
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Manuscript received: December 13, 2016
Revised: February 23, 2017
Final Article published: && &&, &&&&
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Communications
Cell Fixation
Cellfi-Flash: A new fixative for light mi-
croscopy of living cells was obtained by
functionalizing glutaraldehyde with
a photocleavable protecting group. Ester
substituents ensure that the masked
compound can enter the cell and accu-
mulate therein after esterase-mediated
hydrolysis. Incubated cells are instantly
fixed and ready for microscopy upon
irradiation whereas conventional alde-
hyde fixation is diffusion-controlled.
K. M. Schelkle, C. Schmid, K. Yserentant,
M. Bender, I. Wacker, M. Petzoldt,
M. Hamburger, D.-P. Herten,
R. Wombacher, R. R. Schrçder,
U. H. F. Bunz*
&&&& — &&&&
Cell Fixation by Light-Triggered Release of
Glutaraldehyde
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